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Formation of 16-epi-Estriol from Estradiol-17-β in the Laying Hen*
VITAMIN K AND PIGMENTATION
quin or an equimolar level of menadione in the form of menadione sodium bisulfite complex improved pigmentation only slightly. Since continuous high-level feeding of menadione depresses growth, further work is indicated to explore the nature and application of these observations. ACKNOWLEDGMENT The authors gratefully acknowledge the support of Dr. D. F. Green, Merck, Sharp & Dohme in defraying the cost of the color plates. REFERENCES Bunnell, R. H., and J. D. Bauernfeind, 1958. Carotenoids as poultry pigmenters. XI World's Poultry Congress, Section 2, Paper 13. Fritz, J. C , F. D. Wharton, Jr. and L. J. Classen, 1958. Influence of feed on pigmentation of poultry and eggs. X I World's Poultry Congress, Section 3, Paper 14.
Formation of 16-e/>/-Estriol from Estradiol17-/3 in the Laying Hen* H. F. MACRAE AND R. H. COMMON Macdonald College of McGill University, Quebec, Canada (Received for publication September 14, 1959)
M
ACRAE et al. (1959b) injected estradiol 17/3-16-C14 into a laying hen. They were able to demonstrate the presence of radioactive estradiol, estrone and estriol in the gut and gut contents one hour after injection. Hence the fowl, like a number of mammalian species, can convert estradiol into estrone and estriol. They pointed out, however, that much of the radioactivity on their chromatograms was in a fourth spot which they were not at that time in a position to identify. It was possible to show that the spot was not * Contribution from the Faculty of Agriculture, McGill University, Macdonald College, Que., Canada, Journal Series No. 445.
2-methoxy estrone, and it was practically certain that the unstable 16-hydroxyestrones could not have survived the acid hydrolysis and alkali treatments used in extraction. The present paper deals with the subsequent identification of the "fourth spot" as 16-e/>J-estriol. EXPERIMENTAL
One mg. estradiol-17/3-16-C14 (Chas. Frosst and Co., Montreal), containing 2.7 microcuries of radioactivity, was dissolved in 1 ml. propylene glycol and injected into the wing vein of a laying hen. Two hours later the bird was killed by
Downloaded from http://ps.oxfordjournals.org/ at Universiteit van Amsterdam on May 18, 2015
days old. Shank color was then visually scored and the animals sacrificed. A piece of skin was removed from one leg, cleaned of adhering tissue, dried at 80° C. and weighed. Pigments were extracted for 48 hours with a 1:1 mixture of ethanol and chloroform in a mechanical shaker at room temperature. After evaporating the pigment-containing solution to a suitable volume, it was filtered, and read at 450 m/i in a Coleman spectrophotometer. The results were expressed as density readings per gram of dried skin. The visual scoring, the spectrophotometric data and body weights are given in Table 1. The accompanying plate shows the increased pigmentation in the shanks and beak of menadione-fed chicks. It is clear from these data that the menadione supplementation significantly enhanced pigmentation while the antioxidant santo-
707
708
H. F. M A C R A E AND R. H.
A B FIG. 1. Chromatographic separation of estriol and 16-efi-estriol in the system absolute methanollight petroleum at 34°C. Time of running: 6 hours. Stained with DSA. Spot A. 20 micrograms 16-e/>i-estriol Spot B. 20 micrograms 16-e/>s-estriol plus 20 micrograms estriol. NOTE: The estriol gave a pinkish yellow color. The 16-e/>z'-estriol gave a distinctly brownish yellow color.
it was insufficient to give a perceptible stain with diazotized sulphanilic acid (DSA), was mixed with 20 micrograms reference estradiol-17/3 and re-chromatographed. Congruence of stain and radioautogram of the chromatogram established the identity of the zone as estradiol. The presumptive estrone zone was identified as estrone by the same technique, using reference estrone. The foregoing observations confirmed previous findings
Downloaded from http://ps.oxfordjournals.org/ at Universiteit van Amsterdam on May 18, 2015
decapitation. The bile was recovered for separate analysis. The intestinal tract from the gizzard to the cloaca, including contents, was removed and freed from mesenteric fat. The excreta voided since the injection of estradiol were collected and dispersed along with the intestinal tract in methanol by means of a Waring Blendor. The mixture was then worked up for estrogens by the method of Bauld (1957), using acid hydrolysis and including the final partition between water (estriol fraction) and benzene (estrone plus estradiol fraction) but omitting the saponification in N N a O H . The bile was taken u p in methanol and extracted in the same fashion. Chromatography was carried out with a Bush system of absolute methanol plus light petroleum (BP 80°C.-100°C.) at a temperature of 34°C. This system gives its best separation of estrogens when the paper (Whatman No. 1) has been carefully dried in an oven and equilibrated with the solvent system for some hours before the chromatographic run is made (Layne et ak, 1958). Preliminary experimentation confirmed that the system was capable of separating estriol and 16-epiestriol sufficiently sharply (see Fig. 1). The chromatogram of the material in the final benzene phase (the fraction that contains estradiol plus estrone) was examined by radioautography (two weeks exposure). The results indicated the necessity for further chromatography. After two further chromatographic runs in the same system the material was sufficiently purified to yield two distinct radioactive zones with a trace of a third radioactive zone. This third zone corresponded in position with the fourth spot reported by M a c R a e et al. (1959b). The two major zones were then eluted separately with methanol. An aliquot of the eluate from the presumptive estradiol zone, such that
COMMON
FORMATION OF
I
l
1 I
I I
FIG. 2. Radioautograra of chromatogram of the aqueous phase obtained in final stage of the separation, by Bauld's method, of estrogens from a laying hen that had been injected with radioactive estradiol-17/3. Note separation of two distinct dark radioactive zones, with a third faint zone not fully separated from the lower dark zone. The uppermost zone corresponded in position with estriol, and lower dark zone corresponded with 16-e/>i-estriol. The identity of the third faint zone is unknown. Note also that the two dark zones did not differ greatly in intensity.
(MacRae et al., 1959b) in which a different procedure had been used and the benzenewater partition had been omitted. The radioautogram of the chromatogram of the material from the final aqueous phase (the estriol fraction) yielded a broad zone t h a t suggested the presence of two components. This zone was eluted with methanol and chromatographed
709
again. The radioautogram now showed two well-marked zones (Fig. 2). These two zones were eluted separately. An aliquot of the presumptive estriol zone known by a pilot experiment to be insufficient to give perceptible staining with DSA, was mixed with 20 micrograms reference estriol, re-chromatographed, stained with DSA and radioautographed. Congruence of stain and radioautogram confirmed the identification as estriol. The other and less polar zone corresponded clearly to the fourth spot observed previously (MacRae et al., 1959b). Moreover, its separation in the present experiment in the aqueous phase along with estriol suggested that it might be 16-e/>i-estriol. Accordingly, this zone was divided into two halves. One half was eluted with methanol and was chromatographed in the Bush system, stained with DSA and then radioautographed (Fig. 3, Cs and CR). The amount of radioactive material was insufficient to yield a perceptible stain (Fig. 3, Cs). The radioautogram (Fig. 3, CR) displayed a strong zone that corresponded in position with the position of 16-e/>i-estriol together with a faint trace of a less polar radioactive zone. The other half was similarily eluted with methanol solution and the resultant solution was mixed with 20 micrograms inactive reference 16-e£i-estriol (kindly presented by Dr. T.J. Gallagher) and chromatographed (Fig. 3, D s and D R ) . The staining visible on Ds was obviously due entirely to the reference 16-e/>t-estriol (comparison of Cs and Ds, Fig. 3). The congruence of staining and radioautogram (Fig.3,Ds and D R ) confirmed the identification of the radioactive zone as 16-e/>i-estriol. The identity of the very faint, less polar radioactive zone remains uncertain. I t occupied a position approximately the same as would have estradiol-17/3, so that it may have been due to traces of estradiol t h a t had separated with the aqueous phase on
Downloaded from http://ps.oxfordjournals.org/ at Universiteit van Amsterdam on May 18, 2015
I
16-£P/-ESTRIOL
710
H. F. MACRAE AND R. H. COMMON
o
UR
Ds
DR
FIG. 3. Cs.—chromatogram of one half of presumptive 16-e/>j-estriol, stained with DSA. Note absence of perceptible staining. CR.—radioautogram of same strip as Cs. Ds.—chromatogram of other half of presumptive 16-e/>i-estriol plus 20 micrograms reference 16-epiestriol. Stained with DSA. DR.—radioautogram of same strip as Ds. Note congruence of staining of carrier 16-e|>£-estriol and radioautogram of presumptive radioactive 16-e/>i-estriol.
partition between benzene and water. In our previous work we found that the radioactive zone, now shown by finger-printing to be 16-e/>i-estriol, gave a stain with DNPH though not with DSA. The present work shows that this positive reaction with DNPH, which was confusing at the time, must have been due to contaminating material. The negative DSA stain, of course, was a consequence of the minute amounts present. The chromatograms of the extracts of the bile showed the presence of radioactive
estradiol, estrone and estriol, but did not provide any indication of the presence of 16-g/>t-estriol. This confirms the previous observation that the "fourth spot" was absent from radioautograms of bile extracts (MacRae el al., 1959b). DISCUSSION The present work shows that 16-epiestriol is a prominent metabolite of estradiol in the fowl, along with estrone and and estriol. Its presence in the aqueous phase of the Bauld separation, a piece of
Downloaded from http://ps.oxfordjournals.org/ at Universiteit van Amsterdam on May 18, 2015
CS
FORMATION or 16-EPI-ESTRIOL
While the identification of 16-e^i-estriol as a metabolite of estradiol-17/3 in the laying hen is of interest in itself, the present work raises a further point of interest in relation to the pattern of estrogen metabolism in the laying hen as compared to that in the pregnant woman. The ratio estriol: 16-e^'-estriol in preg-
nancy urine has been reported as about 60:1 (Marrian el al., 1957). The estriol is excreted in mgm. quantities and it is easily crystallized from extracts of pregnancy urine. The following evidence, therefore, supports the view that the ratio estriol: 16e^i-estriol in the excreta of the laying hen is much lower than it is in pregnancy urine: (a) The 16-e^'-estriol zones on the radioautograms of our extracts of excreta of the laying hen were darker than any other radioactive zone and darker than the estriol zones: the results, in fact, indicate that 16-e^»i-estriol is a major metabolite of estradiol in the laying hen. (b) We have repeatedly and consistently failed to obtain crystalline estriol from extracts of droppings or of the urine of laying hens in amounts equivalent to 100 bird-days, although chromatographic, spectrometric and chemical evidence showed that estriol was present and although crystalline estradiol-17/3 has been recovered from the droppings of the laying hen (MacRae et al., 1959a). It would appear, therefore, to be more difficult to obtain crystalline estriol from avian droppings and urine than from pregnancy urine. Vacuum sublimation of extracts of large amounts of laying hens' excreta (up to 8 kgm.) yielded only minute amounts of material in the presumptive estriol fraction. These difficulties suggest that the estriol content of laying hens' droppings or urine is relatively small, (c) If the ratio estriol: 16-e/>£-estriol were as high in the excreta of laying hens as in pregnancy urine, it is unlikely that it would have been possible to separate estriol from 16-e£i-estriol so readily in the Bush system as has proved possible in the experiments reported here and previously (MacRae et al., 1959b). (d) Preliminary observations in this laboratory on extracts of the urine of laying hens by Bauld's (1957) method have yielded presumptive
Downloaded from http://ps.oxfordjournals.org/ at Universiteit van Amsterdam on May 18, 2015
evidence that was lacking in our earlier work, provided the first clue to its identity. The fact that a trace of e^i-estriol showed up on the radioautogram of the benzene phase is explained by the observations of Givner (1959), who has shown that 16-e/>j-estriol occurs in both the aqueous and the benzene phases in the Bauld separation of urinary estrogens, although by far the greater proportion is in the aqueous phase. The present work is in agreement with that of Givner (1959) on this point. The metabolic sequence of urinary estrogens in the mammal has been discussed by Marrian (1958) and by Breuer et al. (1959) in the light of various metabolic conversions that have been established in vivo or in vitro or in both ways. Much of the discussion centers on the possible role of 16-ketoestrone. Serchi (1953) reported the presence of 16-ketoestrone in urine and Slaunwhite and Sandberg (1956) detected it in extracts of urine of a normal woman in the luteal phase. Nonetheless, the metabolic and physiological status of this reactive substance is not completely certain. It is known that 16-a-hydroxyestrone can give rise to estriol on incubation with liver slices (Breuer et al., 1959) and it is well-established that estriol can give rise to 16-e^i-estriol in the human subject. There is uncertainty, however, as to whether estriol is transformed in the human subject directly to 16-e^'-estriol or indirectly via 16-ketoestradiol-17/3. Levitz, Spitzer and Twombley (1958) have furnished some evidence in support of the latter view.
711
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H. F. MACRAE AND R. H. COMMON
SUMMARY
1. A laying hen was given an injection of radioactive estradiol-17/3. Subsequent examination of the intestinal tract, together with its contents and the excreta, revealed the presence of radioactive 16e^'-estriol as well as of radioactive estradiol, estrone and estriol. 2. Examination of the bile revealed the presence of radioactive estradiol, estrone and estriol, but radioactive 16-e^'-estriol was not detected in the bile. 3. Evidence is marshalled in support of the view that the ratio estriol: 16-epiestriol in the excreta of the laying hen is considerably lower than the corresponding ratio in human pregnancy urine, and that 16-e/>i-estriol is an important metabolite of estradiol-17/3 in the laying hen. ACKNOWLEDGMENTS
The authors wish to thank the National Research Council of Canada for a grant which defrayed part of the expense of this experiment, and Canadian Industries Ltd., for the Fellowship held by one of us (H. F. MacRae). They wish also to thank Dr. D. A. McGinty, Parke, Davis & Co., Detroit, for presenting a sample of reference estriol, and Dr. T. J. Gallagher,
Sloan-Kettering Institute for Cancer Research, New York, for very kindly presenting a sample of reference 16-e^'-estriol which was essential for this work. They are further indebted to Dr. D.S. Layne, Worcester Research Foundation, Shrewsbury, Mass., and Dr. J. Raeside, Ontario Veterinary College, Guelph, Ont., for many helpful suggestions. REFERENCES Bauld, W., 1957. Chemical determination of estrogens in human urine. Methods of Biochemical Analysis (editor D. Glick), Interscience Publishers Inc., New York. Breuer, H., R. Knupper and G. Pangels, 1959. Stoffwechsel von 16-Ketooestron in menschlichen Geweben. Acta Endocrinologica, 30: 247-258. Givner, M., 1959. Ph.D. Thesis, McGill University, Montreal, Que., Canada. Layne, D. S., R. H. Common, W. A. Maw and R. M. Fraps, 1958. Presence of oestrone, oestradiol and oestriol in extracts of ovaries of laying hens. Nature (London), 181: 351-352. Levitz, M., J. R. Spitzer and G. H. Twombley 1958. Interconversions of 16-oxygenated estrogens. I. The synthesis of estriol-16-C14 and its metabolism in man. J. Biol. Chem. 231: 787— 797. MacRae, H. F., W. Zaharia and R. H. Common, 1959a. Isolation of crystalline estradiol-170 from droppings of laying hens. Poultry Sci. 38: 318-321. MacRae, H. F., D. S. Layne and R. H. Common, 1959b. Formation of estrone, estriol and an unidentified steroid from estradiol in the laying hen. Poultry Sci. 38: 685-687. Marrian, G. F., 1958. The biochemistry of the estrogenic hormones. Proc. Fourth International Congress of Biochemistry Symposium No. IV, Vienna. Marrian, G. F., E. G. D. Watson and M. Panattoni, 1957. The isolation of a ketonic dihydroxy Kober chromogen from the urine of pregnant women. Biochem. J. 650: 12-18. Serchi, G., 1953. The separation of a estratrienic urinary metabolite in crystalline form, by extraction from paper chromatograms. Chimica (Milan), 8: 10-11. (cited from Chem. Abstr. 47: 12496). Slaunwhite, W. R., and A. A. Sandberg, 1956. Studies on the phenolic steroids in human subjects. I. The conversion of 16-C14-estrone to C14-16-ketoestrone. Arch. Biochem. 63:478-480.
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estriol and 16-e/>i-estriol fractions (for one day's urine) of approximately 1 microgram and 3 micrograms respectively. While no claim is made that these amounts give an accurate measurement of the daily excretion of these estrogens by the laying hen, the ratio of the amounts found is considered to be significant. It appears, therefore, that the ratio estriol: 16-e/>«-estriol in the excreta of the laying hen must be very much lower than the ratio in human pregnancy urine, and that this constitutes a difference in the pattern of estrogen excretion as between the pregnant woman and the laying hen.
quin or an equimolar level of menadione in the form of menadione sodium bisulfite complex improved pigmentation only slightly. Since continuous high-level feeding of menadione depresses growth, further work is indicated to explore the nature and application of these observations. ACKNOWLEDGMENT The authors gratefully acknowledge the support of Dr. D. F. Green, Merck, Sharp & Dohme in defraying the cost of the color plates. REFERENCES Bunnell, R. H., and J. D. Bauernfeind, 1958. Carotenoids as poultry pigmenters. XI World's Poultry Congress, Section 2, Paper 13. Fritz, J. C , F. D. Wharton, Jr. and L. J. Classen, 1958. Influence of feed on pigmentation of poultry and eggs. X I World's Poultry Congress, Section 3, Paper 14.
Formation of 16-e/>/-Estriol from Estradiol17-/3 in the Laying Hen* H. F. MACRAE AND R. H. COMMON Macdonald College of McGill University, Quebec, Canada (Received for publication September 14, 1959)
M
ACRAE et al. (1959b) injected estradiol 17/3-16-C14 into a laying hen. They were able to demonstrate the presence of radioactive estradiol, estrone and estriol in the gut and gut contents one hour after injection. Hence the fowl, like a number of mammalian species, can convert estradiol into estrone and estriol. They pointed out, however, that much of the radioactivity on their chromatograms was in a fourth spot which they were not at that time in a position to identify. It was possible to show that the spot was not * Contribution from the Faculty of Agriculture, McGill University, Macdonald College, Que., Canada, Journal Series No. 445.
2-methoxy estrone, and it was practically certain that the unstable 16-hydroxyestrones could not have survived the acid hydrolysis and alkali treatments used in extraction. The present paper deals with the subsequent identification of the "fourth spot" as 16-e/>J-estriol. EXPERIMENTAL
One mg. estradiol-17/3-16-C14 (Chas. Frosst and Co., Montreal), containing 2.7 microcuries of radioactivity, was dissolved in 1 ml. propylene glycol and injected into the wing vein of a laying hen. Two hours later the bird was killed by
Downloaded from http://ps.oxfordjournals.org/ at Universiteit van Amsterdam on May 18, 2015
days old. Shank color was then visually scored and the animals sacrificed. A piece of skin was removed from one leg, cleaned of adhering tissue, dried at 80° C. and weighed. Pigments were extracted for 48 hours with a 1:1 mixture of ethanol and chloroform in a mechanical shaker at room temperature. After evaporating the pigment-containing solution to a suitable volume, it was filtered, and read at 450 m/i in a Coleman spectrophotometer. The results were expressed as density readings per gram of dried skin. The visual scoring, the spectrophotometric data and body weights are given in Table 1. The accompanying plate shows the increased pigmentation in the shanks and beak of menadione-fed chicks. It is clear from these data that the menadione supplementation significantly enhanced pigmentation while the antioxidant santo-
707
708
H. F. M A C R A E AND R. H.
A B FIG. 1. Chromatographic separation of estriol and 16-efi-estriol in the system absolute methanollight petroleum at 34°C. Time of running: 6 hours. Stained with DSA. Spot A. 20 micrograms 16-e/>i-estriol Spot B. 20 micrograms 16-e/>s-estriol plus 20 micrograms estriol. NOTE: The estriol gave a pinkish yellow color. The 16-e/>z'-estriol gave a distinctly brownish yellow color.
it was insufficient to give a perceptible stain with diazotized sulphanilic acid (DSA), was mixed with 20 micrograms reference estradiol-17/3 and re-chromatographed. Congruence of stain and radioautogram of the chromatogram established the identity of the zone as estradiol. The presumptive estrone zone was identified as estrone by the same technique, using reference estrone. The foregoing observations confirmed previous findings
Downloaded from http://ps.oxfordjournals.org/ at Universiteit van Amsterdam on May 18, 2015
decapitation. The bile was recovered for separate analysis. The intestinal tract from the gizzard to the cloaca, including contents, was removed and freed from mesenteric fat. The excreta voided since the injection of estradiol were collected and dispersed along with the intestinal tract in methanol by means of a Waring Blendor. The mixture was then worked up for estrogens by the method of Bauld (1957), using acid hydrolysis and including the final partition between water (estriol fraction) and benzene (estrone plus estradiol fraction) but omitting the saponification in N N a O H . The bile was taken u p in methanol and extracted in the same fashion. Chromatography was carried out with a Bush system of absolute methanol plus light petroleum (BP 80°C.-100°C.) at a temperature of 34°C. This system gives its best separation of estrogens when the paper (Whatman No. 1) has been carefully dried in an oven and equilibrated with the solvent system for some hours before the chromatographic run is made (Layne et ak, 1958). Preliminary experimentation confirmed that the system was capable of separating estriol and 16-epiestriol sufficiently sharply (see Fig. 1). The chromatogram of the material in the final benzene phase (the fraction that contains estradiol plus estrone) was examined by radioautography (two weeks exposure). The results indicated the necessity for further chromatography. After two further chromatographic runs in the same system the material was sufficiently purified to yield two distinct radioactive zones with a trace of a third radioactive zone. This third zone corresponded in position with the fourth spot reported by M a c R a e et al. (1959b). The two major zones were then eluted separately with methanol. An aliquot of the eluate from the presumptive estradiol zone, such that
COMMON
FORMATION OF
I
l
1 I
I I
FIG. 2. Radioautograra of chromatogram of the aqueous phase obtained in final stage of the separation, by Bauld's method, of estrogens from a laying hen that had been injected with radioactive estradiol-17/3. Note separation of two distinct dark radioactive zones, with a third faint zone not fully separated from the lower dark zone. The uppermost zone corresponded in position with estriol, and lower dark zone corresponded with 16-e/>i-estriol. The identity of the third faint zone is unknown. Note also that the two dark zones did not differ greatly in intensity.
(MacRae et al., 1959b) in which a different procedure had been used and the benzenewater partition had been omitted. The radioautogram of the chromatogram of the material from the final aqueous phase (the estriol fraction) yielded a broad zone t h a t suggested the presence of two components. This zone was eluted with methanol and chromatographed
709
again. The radioautogram now showed two well-marked zones (Fig. 2). These two zones were eluted separately. An aliquot of the presumptive estriol zone known by a pilot experiment to be insufficient to give perceptible staining with DSA, was mixed with 20 micrograms reference estriol, re-chromatographed, stained with DSA and radioautographed. Congruence of stain and radioautogram confirmed the identification as estriol. The other and less polar zone corresponded clearly to the fourth spot observed previously (MacRae et al., 1959b). Moreover, its separation in the present experiment in the aqueous phase along with estriol suggested that it might be 16-e/>i-estriol. Accordingly, this zone was divided into two halves. One half was eluted with methanol and was chromatographed in the Bush system, stained with DSA and then radioautographed (Fig. 3, Cs and CR). The amount of radioactive material was insufficient to yield a perceptible stain (Fig. 3, Cs). The radioautogram (Fig. 3, CR) displayed a strong zone that corresponded in position with the position of 16-e/>i-estriol together with a faint trace of a less polar radioactive zone. The other half was similarily eluted with methanol solution and the resultant solution was mixed with 20 micrograms inactive reference 16-e£i-estriol (kindly presented by Dr. T.J. Gallagher) and chromatographed (Fig. 3, D s and D R ) . The staining visible on Ds was obviously due entirely to the reference 16-e/>t-estriol (comparison of Cs and Ds, Fig. 3). The congruence of staining and radioautogram (Fig.3,Ds and D R ) confirmed the identification of the radioactive zone as 16-e/>i-estriol. The identity of the very faint, less polar radioactive zone remains uncertain. I t occupied a position approximately the same as would have estradiol-17/3, so that it may have been due to traces of estradiol t h a t had separated with the aqueous phase on
Downloaded from http://ps.oxfordjournals.org/ at Universiteit van Amsterdam on May 18, 2015
I
16-£P/-ESTRIOL
710
H. F. MACRAE AND R. H. COMMON
o
UR
Ds
DR
FIG. 3. Cs.—chromatogram of one half of presumptive 16-e/>j-estriol, stained with DSA. Note absence of perceptible staining. CR.—radioautogram of same strip as Cs. Ds.—chromatogram of other half of presumptive 16-e/>i-estriol plus 20 micrograms reference 16-epiestriol. Stained with DSA. DR.—radioautogram of same strip as Ds. Note congruence of staining of carrier 16-e|>£-estriol and radioautogram of presumptive radioactive 16-e/>i-estriol.
partition between benzene and water. In our previous work we found that the radioactive zone, now shown by finger-printing to be 16-e/>i-estriol, gave a stain with DNPH though not with DSA. The present work shows that this positive reaction with DNPH, which was confusing at the time, must have been due to contaminating material. The negative DSA stain, of course, was a consequence of the minute amounts present. The chromatograms of the extracts of the bile showed the presence of radioactive
estradiol, estrone and estriol, but did not provide any indication of the presence of 16-g/>t-estriol. This confirms the previous observation that the "fourth spot" was absent from radioautograms of bile extracts (MacRae el al., 1959b). DISCUSSION The present work shows that 16-epiestriol is a prominent metabolite of estradiol in the fowl, along with estrone and and estriol. Its presence in the aqueous phase of the Bauld separation, a piece of
Downloaded from http://ps.oxfordjournals.org/ at Universiteit van Amsterdam on May 18, 2015
CS
FORMATION or 16-EPI-ESTRIOL
While the identification of 16-e^i-estriol as a metabolite of estradiol-17/3 in the laying hen is of interest in itself, the present work raises a further point of interest in relation to the pattern of estrogen metabolism in the laying hen as compared to that in the pregnant woman. The ratio estriol: 16-e^'-estriol in preg-
nancy urine has been reported as about 60:1 (Marrian el al., 1957). The estriol is excreted in mgm. quantities and it is easily crystallized from extracts of pregnancy urine. The following evidence, therefore, supports the view that the ratio estriol: 16e^i-estriol in the excreta of the laying hen is much lower than it is in pregnancy urine: (a) The 16-e^'-estriol zones on the radioautograms of our extracts of excreta of the laying hen were darker than any other radioactive zone and darker than the estriol zones: the results, in fact, indicate that 16-e^»i-estriol is a major metabolite of estradiol in the laying hen. (b) We have repeatedly and consistently failed to obtain crystalline estriol from extracts of droppings or of the urine of laying hens in amounts equivalent to 100 bird-days, although chromatographic, spectrometric and chemical evidence showed that estriol was present and although crystalline estradiol-17/3 has been recovered from the droppings of the laying hen (MacRae et al., 1959a). It would appear, therefore, to be more difficult to obtain crystalline estriol from avian droppings and urine than from pregnancy urine. Vacuum sublimation of extracts of large amounts of laying hens' excreta (up to 8 kgm.) yielded only minute amounts of material in the presumptive estriol fraction. These difficulties suggest that the estriol content of laying hens' droppings or urine is relatively small, (c) If the ratio estriol: 16-e/>£-estriol were as high in the excreta of laying hens as in pregnancy urine, it is unlikely that it would have been possible to separate estriol from 16-e£i-estriol so readily in the Bush system as has proved possible in the experiments reported here and previously (MacRae et al., 1959b). (d) Preliminary observations in this laboratory on extracts of the urine of laying hens by Bauld's (1957) method have yielded presumptive
Downloaded from http://ps.oxfordjournals.org/ at Universiteit van Amsterdam on May 18, 2015
evidence that was lacking in our earlier work, provided the first clue to its identity. The fact that a trace of e^i-estriol showed up on the radioautogram of the benzene phase is explained by the observations of Givner (1959), who has shown that 16-e/>j-estriol occurs in both the aqueous and the benzene phases in the Bauld separation of urinary estrogens, although by far the greater proportion is in the aqueous phase. The present work is in agreement with that of Givner (1959) on this point. The metabolic sequence of urinary estrogens in the mammal has been discussed by Marrian (1958) and by Breuer et al. (1959) in the light of various metabolic conversions that have been established in vivo or in vitro or in both ways. Much of the discussion centers on the possible role of 16-ketoestrone. Serchi (1953) reported the presence of 16-ketoestrone in urine and Slaunwhite and Sandberg (1956) detected it in extracts of urine of a normal woman in the luteal phase. Nonetheless, the metabolic and physiological status of this reactive substance is not completely certain. It is known that 16-a-hydroxyestrone can give rise to estriol on incubation with liver slices (Breuer et al., 1959) and it is well-established that estriol can give rise to 16-e^i-estriol in the human subject. There is uncertainty, however, as to whether estriol is transformed in the human subject directly to 16-e^'-estriol or indirectly via 16-ketoestradiol-17/3. Levitz, Spitzer and Twombley (1958) have furnished some evidence in support of the latter view.
711
712
H. F. MACRAE AND R. H. COMMON
SUMMARY
1. A laying hen was given an injection of radioactive estradiol-17/3. Subsequent examination of the intestinal tract, together with its contents and the excreta, revealed the presence of radioactive 16e^'-estriol as well as of radioactive estradiol, estrone and estriol. 2. Examination of the bile revealed the presence of radioactive estradiol, estrone and estriol, but radioactive 16-e^'-estriol was not detected in the bile. 3. Evidence is marshalled in support of the view that the ratio estriol: 16-epiestriol in the excreta of the laying hen is considerably lower than the corresponding ratio in human pregnancy urine, and that 16-e/>i-estriol is an important metabolite of estradiol-17/3 in the laying hen. ACKNOWLEDGMENTS
The authors wish to thank the National Research Council of Canada for a grant which defrayed part of the expense of this experiment, and Canadian Industries Ltd., for the Fellowship held by one of us (H. F. MacRae). They wish also to thank Dr. D. A. McGinty, Parke, Davis & Co., Detroit, for presenting a sample of reference estriol, and Dr. T. J. Gallagher,
Sloan-Kettering Institute for Cancer Research, New York, for very kindly presenting a sample of reference 16-e^'-estriol which was essential for this work. They are further indebted to Dr. D.S. Layne, Worcester Research Foundation, Shrewsbury, Mass., and Dr. J. Raeside, Ontario Veterinary College, Guelph, Ont., for many helpful suggestions. REFERENCES Bauld, W., 1957. Chemical determination of estrogens in human urine. Methods of Biochemical Analysis (editor D. Glick), Interscience Publishers Inc., New York. Breuer, H., R. Knupper and G. Pangels, 1959. Stoffwechsel von 16-Ketooestron in menschlichen Geweben. Acta Endocrinologica, 30: 247-258. Givner, M., 1959. Ph.D. Thesis, McGill University, Montreal, Que., Canada. Layne, D. S., R. H. Common, W. A. Maw and R. M. Fraps, 1958. Presence of oestrone, oestradiol and oestriol in extracts of ovaries of laying hens. Nature (London), 181: 351-352. Levitz, M., J. R. Spitzer and G. H. Twombley 1958. Interconversions of 16-oxygenated estrogens. I. The synthesis of estriol-16-C14 and its metabolism in man. J. Biol. Chem. 231: 787— 797. MacRae, H. F., W. Zaharia and R. H. Common, 1959a. Isolation of crystalline estradiol-170 from droppings of laying hens. Poultry Sci. 38: 318-321. MacRae, H. F., D. S. Layne and R. H. Common, 1959b. Formation of estrone, estriol and an unidentified steroid from estradiol in the laying hen. Poultry Sci. 38: 685-687. Marrian, G. F., 1958. The biochemistry of the estrogenic hormones. Proc. Fourth International Congress of Biochemistry Symposium No. IV, Vienna. Marrian, G. F., E. G. D. Watson and M. Panattoni, 1957. The isolation of a ketonic dihydroxy Kober chromogen from the urine of pregnant women. Biochem. J. 650: 12-18. Serchi, G., 1953. The separation of a estratrienic urinary metabolite in crystalline form, by extraction from paper chromatograms. Chimica (Milan), 8: 10-11. (cited from Chem. Abstr. 47: 12496). Slaunwhite, W. R., and A. A. Sandberg, 1956. Studies on the phenolic steroids in human subjects. I. The conversion of 16-C14-estrone to C14-16-ketoestrone. Arch. Biochem. 63:478-480.
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estriol and 16-e/>i-estriol fractions (for one day's urine) of approximately 1 microgram and 3 micrograms respectively. While no claim is made that these amounts give an accurate measurement of the daily excretion of these estrogens by the laying hen, the ratio of the amounts found is considered to be significant. It appears, therefore, that the ratio estriol: 16-e/>«-estriol in the excreta of the laying hen must be very much lower than the ratio in human pregnancy urine, and that this constitutes a difference in the pattern of estrogen excretion as between the pregnant woman and the laying hen.